PROJECT SUMMARY Drug addiction is a complex and devastating disease characterized by compulsive drug seeking and use, despite harmful consequences. This urgent social and health problem contributes to 90,000 deaths per year and an annual cost of over $700 billion in the United States alone (see NIDA website). Despite its complexity, it is generally believed that long-term maladaptive changes in the mesolimbic dopamine (DA) reward system play a central role in the progression from casual to compulsive drug use and to contribute to the high rates of relapse in human addicts. Indeed, while extensive efforts have aimed to achieve abstinence, understanding the molecular mechanisms underlying drug relapse is critical for maintaining a drug-free state. Accordingly, accumulating evidence suggests that drug-induced epigenetic and chromatin changes play an important role in mediating addiction-related behavior, but due to technical hurdles in dealing with cellular heterogeneity of the mammalian brain, most of the molecular studies so far have been performed using mixed cell populations, thus confounding data interpretation. To address this issue, I have led the effort to develop a robust in vivo nuclear tagging system with neuron subtype specificity that facilitates transcriptome and chromatin accessibility profiling. Accessible chromatin profiles are cell-type specific and can be used to identify regulatory regions such as enhancers of active genes. This proposal therefore seeks to understand the role of dopaminergic enhancers during cocaine craving in order to regulate relapse to cocaine-seeking. I hypothesize that enhancer elements play an important role in regulating gene expression changes induced by cocaine craving, and that these changes can affect the likelihood and magnitude of relapse to cocaine-seeking. To test this hypothesis, I propose to profile chromatin accessibility and gene expression changes associated with cocaine craving in DA neurons, using a mouse model of intravenous cocaine self-administration (IVSA). The epigenomics and bioinformatics training I will receive during this K01 Award will allow me to reveal candidate genes and molecular pathways specific to this phase of addiction. Further, using CRISPR/Cas9 genome editing techniques, I will edit top craving- associated enhancers in DA neurons to validate their ability to regulate transcriptional activity in vivo. Finally, I will test the functional role of these enhancers in regulating cocaine relapse behavior by IVSA. Completion of the proposed studies will not only advance our understanding of molecular mechanisms underlying cocaine addiction and identify novel therapeutic targets, but will also provide a novel, broadly applicable strategy for manipulating and understanding epigenetic regulation in brain with cell-type specificity.